2023 White Paper on Recent Issues in Bioanalysis: EU IVDR 2017/746 Implementation/Impact, IVD/CDx/CLIA Approved Assays, High Dimensional Cytometry, Multiplexing Technologies, LBA Tissue Analysis, Vaccine Study Endpoints, Cell-Based Assays for Biomarkers, Cell Therapy and Vaccines ( - Recommendations on Development & Validation of Biomarkers, IVD, CDx, Cell-Based, Flow Cytometry, Ligand-Binding and Enzyme Assays; Advanced Critical Reagents Strategies).

The 17 Workshop on Recent Issues in Bioanalysis (17 WRIB) took place in Orlando, FL, USA on 19-23 June 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17 WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.

Systematic optimization of targeted and multiplexed MS-based screening workflows for protein biomarkers.

The capability of targeted MS-based methods to simultaneously measure multiple analytes with high selectivity and sensitivity greatly facilitates the discovery and quantitation of novel biomarkers. However, the complexity of biological samples is a major bottleneck that requires extensive sample preparation. This paper reports a generic workflow to optimize surrogate peptide-based protein biomarker screening for seven human proteins in a multiplexed manner without the need for any specific affinity reagents.

Identification of hnRNP-A1 as a pharmacodynamic biomarker of type I PRMT inhibition in blood and tumor tissues.

Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development.

Intact mAb LC-MS for drug concentration from pre-clinical studies: bioanalytical method performance and in-life samples.

Antibody biotherapeutic measurement from pharmacokinetic studies has not been traditionally based on intact molecular mass as is the case for small molecules. However, recent advancements in protein capture and mass spectrometer technology have enabled intact mass detection and quantitation for dosed biotherapeutics. A bioanalytical method validation is part of the regulatory requirement for sample analysis to determine drug concentration from in-life study samples.

Antibody Subunit LC-MS Analysis for Pharmacokinetic and Biotransformation Determination from In-Life Studies for Complex Biotherapeutics.

Complex biotherapeutics present challenges from drug discovery, screening, and development perspectives. While monoclonal antibody drugs are not monitored for metabolites in the same manner as small molecules, biotherapeutics such as fusion proteins, antibody-drug conjugates, or bispecific antibodies may undergo biotransformation (such as clipping, deamidation, or oxidation) in vivo, resulting in catabolites that can have a direct impact on drug safety or efficacy. Here antibody subunit LC-MS is utilized for evaluation of two classes of complex biotherapeutics: an antibody-drug conjugate and a mAb-fusion biotherapeutic.

2019 White Paper on Recent Issues in Bioanalysis: Chromatographic Assays (Part 1 - Innovation in Small Molecules and Oligonucleotides & Mass Spectrometric Method Development Strategies for Large Molecule Bioanalysis).

The 2019 13 Workshop on Recent Issues in Bioanalysis (WRIB) took place in New Orleans, LA, USA on April 1-5, 2019 with an attendance of over 1000 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers, immunogenicity and gene therapy. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS, LBA cell-based/flow cytometry assays and qPCR approaches.

Microflow UPLC and high-resolution MS as a sensitive and robust platform for quantitation of intact peptide hormones.

Recent advances in microflow ultra performance liquid chromatography (UPLC) systems offer higher sensitivity with robustness to meet the routine bioanalytical demands. Modern high-resolution mass spectrometers (HRMS) enable the development of highly selective methods with broad dynamic range. The quantitative performances of tandem quadrupole MS and HRMS were comprehensively compared using seven intact peptide hormones up to 9.

Improved Sensitivity for Protein Turnover Quantification by Monitoring Immonium Ion Isotopologue Abundance.

We describe an analytical strategy allowing for the direct quantification of stable isotope label incorporation in newly synthesized proteins following administration of the stable isotope tracer deuterium oxide. We present a demonstration of coupling high-resolution mass spectrometry, metabolic stable isotope labeling, and MS/MS-based isotopologue quantification for the measurement of protein turnover. Stable isotope labeling with deuterium oxide, followed by immonium ion isotopologue quantification, is a more sensitive strategy for determining protein fractional synthesis rates compared to peptide centric mass isotopomer distribution analysis approaches when labeling time and/or stable isotope tracer exposure is limited and, as such, offers a great advantage for human studies.

2018 White Paper on Recent Issues in Bioanalysis: focus on immunogenicity assays by hybrid LBA/LCMS and regulatory feedback (Part 2 - PK, PD & ADA assays by hybrid LBA/LCMS & regulatory agencies' inputs on bioanalysis, biomarkers and immunogenicity).

The 2018 12 Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches.

2017 White Paper: rise of hybrid LBA/LCMS immunogenicity assays (Part 2: hybrid LBA/LCMS biotherapeutics, biomarkers & immunogenicity assays and regulatory agencies' inputs).

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California on 3-7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid ligand binding assay (LBA)/LCMS and LBA approaches.

2017 White Paper on recent issues in bioanalysis: aren't BMV guidance/guidelines 'Scientific'? (Part 1 - LCMS: small molecules, peptides and small molecule biomarkers).

The 2017 11th Workshop on Recent Issues in Bioanalysis (11th WRIB) took place in Los Angeles/Universal City, California from 3 April 2017 to 7 April 2017 with participation of close to 750 professionals from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, weeklong event - A Full Immersion Week of Bioanalysis, Biomarkers and Immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small and large molecule analysis involving LCMS, hybrid LBA/LCMS and ligand-binding assay (LBA) approaches.

Ocular tissue distribution and pharmacokinetic study of a small 13kDa domain antibody after intravitreal, subconjuctival and eye drop administration in rabbits.

Domain antibodies (dAb's) comprise the smallest functional unit of human IgG and can be targeted to a range of different soluble cytokine and receptor targets in the eye. In particular their small size may offer advantage for ocular tissue penetration and distribution. To investigate this we used a 13kDa tool molecule to undertake a preliminary short term ocular tissue distribution and pharmacokinetic study in the rabbit eye.

Microvilli Morphology Can Affect Efflux Active P-Glycoprotein in Confluent MDCKII -hMDR1-NKI and Caco-2 Cell Monolayers.

From fits of drug transport kinetics across confluent MDCKII-hMDR1-NKI and Caco-2 cell monolayers we estimated the levels of efflux active P-glycoprotein (P-gp) in these two cell lines (companion paper). In the present work, we compared the efflux active P-gp number to the total P-gp level, using liquid chromatography-tandem mass spectrometry, and showed that in Caco-2 cells total P-gp is about 10-fold greater than efflux active P-gp, whereas in MDCKII-hMDR1-NKI cells these values are within twofold. We further visualized the microvilli in MDCKII-hMDR1-NKI and Caco-2 cells using three-dimensional structured illumination super-resolution microscopy and found that the microvilli in Caco-2 cells are taller and more densely packed than those in MDCK-hMDR1-NKI cells.

Application of high-resolution MS for development of peptide and large-molecule drug candidates.

Background: For quantitative bioanalysis utilizing MS, the instrument of choice is typically a triple quadruple mass spectrometer. However, advances in high-resolution MS have allowed sensitivity and dynamic ranges to approach that of triple quadrupole instruments.

Results: A matrix-free protein digest, a digested therapeutic protein and the intact peptide therapeutic liraglutide were each analyzed on high-resolution and triple quadrupole mass spectrometers with data compared.

Utilizing enzymatic digestion procedures in the bioanalytical laboratory.

In recent years, the use of LC-MS technologies in the bioanlytical laboratory for quantitation of peptide/protein biomarkers and biotherapeutics has increased dramatically. The increased interest is due to the improvement in sensitivity of MS instruments over the last 5-10 years, as well as its proven ability to overcome some common issues associated with immunoassay, namely selectivity and reagent availability. However, large proteins (>10 kDa) chromatograph and ionize poorly.

2015 White Paper on recent issues in bioanalysis: focus on new technologies and biomarkers (Part 2 - hybrid LBA/LCMS and input from regulatory agencies).

The 2015 9th Workshop on Recent Issues in Bioanalysis (9th WRIB) took place in Miami, Florida with participation of over 600 professionals from pharmaceutical and biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. It is once again a 5-day week long event - a full immersion bioanalytical week - specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches including the focus on biomarkers and immunogenicity.

Camphanic acid chloride: a powerful derivatization reagent for stereoisomeric separation and its DMPK applications.

Background: Camphanic acid chloride has proven to be an efficient chiral derivatization reagent for determination of stereoisomers.

Results: The utility of chemical derivatization of various stereoisomers containing hydroxy functional groups with camphanic acid chloride in the presence or absence of a base is highlighted. This procedure is shown to be relatively simple, fast and a cost-effective method of separating racemic drugs and stereoisomeric metabolites in biological matrices.

2014 White Paper on recent issues in bioanalysis: a full immersion in bioanalysis (Part 2 - hybrid LBA/LCMS, ELN & regulatory agencies' input).

The 2014 8th Workshop on Recent Issues in Bioanalysis (8th WRIB), a 5-day full immersion in the evolving field of bioanalysis, took place in Universal City, California, USA. Close to 500 professionals from pharmaceutical and biopharmaceutical companies, contract research organizations and regulatory agencies worldwide convened to share, review, discuss and agree on approaches to address current issues of interest in bioanalysis. The topics covered included both small and large molecules, and involved LCMS, hybrid LBA/LCMS, LBA approaches and immunogenicity.

Integrating internal and external bioanalytical support to deliver a diversified pharmaceutical portfolio.

The portfolios of pharmaceutical companies have diversified substantially over recent years in recognition that monotherapies and/or small molecules are less suitable for modulating many complex disease etiologies. Furthermore, there has been increased pressure on drug-development budgets over this same period. This has placed new challenges in the path of bioanalytical scientists, both within the industry and with contract research organizations (CROs).

The Gyrolab™ immunoassay system: a platform for automated bioanalysis and rapid sample turnaround.

Background: The Gyrolab™ workstation benefits from fully automated transfer of reagents and samples originating from a storage microplate onto a compact disc containing solid-phase microstructures composed of a 15 nl streptavidin-derivitized bead bed.

Results: This paper describes the development, full validation and use of the method in a regulated environment to measure a humanized bispecific monoclonal antibody-domain antibody (GSK-A) molecule using the Gyrolab immunoassay system in cynomolgus nonhuman primate plasma ranging from 5 to 250 µg/ml. The method was subsequently used in support of the TK portion of a regulated preclinical study in monkeys.

Comparison of the quantification of a therapeutic protein using nominal and accurate mass MS/MS.

Background: The quantification of proteins and peptides in in vivo samples is a critical part of supporting the drug development process for biotherapeutics. LC-MS/MS using tandem quadrupole mass spectrometers is well established as the technology of choice for the quantification of small-molecule drugs and their metabolites in biological fluid. The application of accurate mass MS for quantification in a DMPK environment has attracted considerable interest in recent years.